identification-footprints-rnaprotein-complexes-via-rna

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

The Ohio State University

During messenger RNA (mRNA) biogenesis and processing in the nucleus, many proteins are imprinted on mRNAs assembling them into messenger ribonucleoproteins (mRNPs). Some of these proteins remain stably bound within mRNPs and have a long-lasting impact on their fate. One of the best-studied examples is the exon junction complex (EJC), a multiprotein complex deposited primarily 24 nucleotides upstream of exon-exon junctions as a consequence of pre-mRNA splicing. The EJC maintains a stable, sequence-independent, hold on the mRNA until its removal during translation in the cytoplasm. Acting as a molecular shepherd, the EJC travels with mRNA across the cellular landscape coupling pre-mRNA splicing to downstream, posttranscriptional processes such as mRNA export, mRNA localization, translation, and nonsense-mediated mRNA decay (NMD). In this review, we discuss our current understanding of the EJC's functions during these processes, and expound its newly discovered functions (e.g., pre-mRNA splicing). Another focal point is the recently unveiled in vivo EJC interactome, which has shed new light on the EJC's location on the spliced RNAs and its intimate relationship with other mRNP components. We summarize new strides being made in connecting the EJC's molecular function with phenotypes, informed by studies of human disorders and model organisms. The progress toward understanding EJC functions has revealed, in its wake, even more questions, which are discussed throughout. WIREs RNA 2017, 8:e1411. doi: 10.1002/wrna.1411 For further resources related to this article, please visit the WIREs website.

The exon junction complex: a lifelong guardian of mRNA fate.

The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition, and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. We show that the EJC originates as an SR-rich mega-dalton-sized RNP that contains RNPS1 but lacks CASC3. Sometime before or during translation, the EJC undergoes compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general, whereas CASC3 is needed for NMD of only select mRNAs. The switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD.

Lauren Woodward

Department of Molecular Genetics,
Center for RNA Biology,
Department of Molecular Genetics, Center for RNA Biology

The Ohio State University

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Lauren Woodward

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Genetics,Center for RNA Biology,Department of Molecular Genetics, Center for RNA Biology

The Ohio State University

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Department of Molecular Genetics,
Center for RNA Biology,
Department of Molecular Genetics, Center for RNA Biology